Background: Today’s pharmaceutical markets are crowded with antibacterial agents, yet their toxicities and resistance are always contentious. Azadirachta indica is used in India as an age-old treatment for diverse medical conditions. This study was put forth to analyze the antibacterial action of A. indica bark extract on different bacterial pathogens. Materials and Methods:A. indica bark extract was prepared using Soxhlet extraction method. The bark extract of A. indica was screened for antibacterial activity by agar well diffusion assay technique against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus mirabilis. Ciprofloxacin (5 µg per disc) was used as positive control. All assays were carried out under strict aseptic precautions. All the concentrations included were duplicated, and the results obtained were the average of two independent experiments. Broth microdilution method was used to study the minimum inhibitory concentration of the bark extract of A. indica. Results:A. indica bark extract has shown antibacterial activity against all concentrations of bark extract, whereas antibacterial activity against S. aureus was observed at higher concentration of >500 µg/mL. The bark extract of A. indica showed zone of clearance against E. faecalis, P. aeruginosa, and P. mirabilis at all concentrations. Zone of inhibition was observed in higher concentrations against S. aureus. Conclusion: This study has shown that A. indica bark extract has a potent antibacterial property against S. aureus, E. faecalis, P. aeruginosa, and P. mirabilis. Further isolation of bioactive compounds may lead to a new scope in the treatment of bacterial infections.

Azadirachta indica is a member of Mahogany family. It is a sacred gift of nature, grown majorly in the Indian subcontinent. The Latinized name of neem is “Azadirachta indica,” Azad means “free,” dirakht means “tree,” and i-Hind means “of Indian origin.” Thus, it literally means “The free tree of India.”[1] Neem is used in India from time immemorial for small pox and other infectious diseases. Thousands of years ago, the earliest mention about the uses of neem were scripted in Thirumular Thirumantiram Ennayiram, Tholkappium, and the ancient Tamil works of Sangam literature.[2] Neem contains phytochemical constituents, which exert antiviral, antibacterial, antimalarial, anti-ulcer, and contraceptive properties.[3],[4],[5] The bark of the neem contains tannin-like substance that inhibits bacterial growth.[6]

Gram-positive and gram-negative organisms are more prone to cause maladies such as anthrax, rheumatic fever, diphtheria, pharyngitis, cholera, typhoid fever, shigellosis, Salmonellosis More Details, and whooping cough. Synthetic antibacterial agents are effective against various pathogens, but the major constraints of use are their adverse effects. Medical plants have tremendous contribution in new drug discoveries which has been the plinth for researches in medicinal plants all over the world. The primary benefits of using plant-derived medicines are high-safety margin when compared with synthetic derivatives. Plant-derived medicines also offer profound efficacy at an affordable cost. Current research in medicine shows a defined move toward the use of natural remedies, such as plant products, deviating itself from chemotherapeutic regimens.[7] The indiscriminate use of antimicrobial drugs has paved its path to multiple drug resistance among human pathogenic organisms.[8] Antibiotic resistance has become a global concern, and the search for a newer, safer alternative to existing drugs is the prime option. Thus, this study was postulated to analyze the antibacterial role of A. indica bark extract.

Materials and Methods

Preparation of A. indica crude bark extract

The bark of A. indica was peeled from the stem using a sharp knife and chopped into pieces. Then it was dried and ground into powder. The extract was prepared using Soxhlet extraction of 20mg of each sample for 6h in approximately 250mL methanol and then concentrated to dryness under reduced pressure using rotatory evaporator, and the residues were stored at 4°C.[9]

Microorganisms

Test samples were collected from 120 patients, of which 64 were culture positive and 56 were culture negative. The bacterial pathogens were isolated from various clinical specimens: urine, sputum, blood, and wound infection. Conventional biochemical methods were used for bacterial identification in accordance with the standard microbiological techniques. In this study, the bacterial test strains used were Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus mirabilis. The bacterial strains were selected as two gram-positive and two gram-negative organisms to find out the gram-positive and gram-negative coverage of A. indica bark extract. The strains were maintained in Mueller Hinton Agar, Hi-media laboratories Ltd, Mumbai, India. (MHA, pH 7.2) at 37±1°C. The stock culture slant was maintained at 4°C.

Screening of antimicrobial activity

The bark extract of A. indica was screened for antibacterial activity by agar well diffusion assay technique.[10] Microbe culture (500 µL) grown for 1824h was added to petri plates and molten MHA at 45°C was poured. After the solidification of the agar, appropriate wells were made on the agar surface by using a 5-mm sterile cork borer. The bark extract of A. indica was made into different concentrations by using dimethyl sulfoxide (DMSO). Fifty microliter of each concentration was added in the wells. The inoculated agar plates were left in the refrigerator for approximately 1h for proper diffusion. Then the bacterial cultures were incubated at 37°C for 24h. Ciprofloxacin (5 µg per disc) was used as the positive control. All assays were carried out under strict aseptic precautions. All the concentrations included were duplicated, and the results obtained were the average of two independent experiments.

Determination of minimum inhibitory concentration

Broth microdilution method was used to study the minimum inhibitory concentration (MIC) of the bark extract of A. indica.[11] Twofold dilution series of extract was prepared (500, 1000, and 2000 µg/mL) in microtiter wells. After incubation at 37°C for 24h, microtiter wells were observed for any visible growth. Bacterial suspensions were used as positive control and extracts in the broth were used as negative control. Interpretation of MIC was done as the lowest concentration of the extract that did not show any visible growth on comparing with the control tube.

Results

All the four strains of bacteria showed sensitivity to ciprofloxacin. A. indica bark extract has shown antibacterial activity against all concentrations of bark extracts, whereas antibacterial activity against S. aureus was observed at higher concentration of >500 µg/mL. [Graph 1] shows the result of agar well diffusion method. The MIC was tested for the bark extract of A. indica, which showed zone of clearance against E. faecalis, P. aeruginosa, and P. mirabilis at all concentrations. Although the bark extract did not show significant effect at a low concentration of 500 µg/mL against S. aureus, higher concentrations of 1000 and 2000 µg/mL showed zone of inhibition against S. aureus. [Graph 2] shows the mean zone of inhibition for different concentrations of A. indica bark extracts. DMSO was used as a negative control, and it did not show any zone of inhibition against all the four bacterial strains.

A. indica commonly called as neem all over India retains numerous therapeutic properties. The most important active constituents in A. indica are nimbolinin, gedunin, nimbidin, salannin, quercetin, nimbidol, polyphenolic flavonoids, and ascorbic acid.[12],[13] Because of their special property of possessing multiple ingredients, they show multifarious therapeutic actions. [Figure 1] shows the various therapeutic effects of neem. A study by Ghimeray et al.[14] showed that A. indica bark extract grown in the foothills had significant antioxidant properties. A study by Arora et al.[15] and Biswas et al.[16] showed that bark and leaf extract of neem possessed immunomodulatory effects. The exact molecular mechanism for antibacterial property is not clearly defined, it is considered to be due to its rich antioxidant nature and anti-inflammatory properties.

A. indica also plays a hepatoprotective role. A study by Baligar et al.[17] analyzed the hepatoprotective role of A. indica in carbon tetrachloride–induced hepatotoxicity in rats. Histology and ultrastructure results confirmed that pretreatment with A. indica dose dependently reduced hepatocellular necrosis.[17] Current antimicrobial agents such as amoxicillin clavulanate, flucloxacillin, nitrofurantoin, erythromycin, sulfonamide, sulfasalazine, and telithromycin are more prone for hepatotoxicity.[18]A. indica is both hepatoprotective and antibacterial; hence, it can be used as a synergistic agent with the hepatotoxic antimicrobial agents for better spectrum of coverage. It can be safely used for patients with hepatic failure.

A study by Yerima et al.[19] showed antibacterial activity of bark, seed, leaf, and fruit extracts of A. indica against bacteria isolated from adult mouth. The phytochemical compound of A. indica showed strong antibacterial activity in a study by Pennington et al.[20] In this study, neem bark extracts have shown antibacterial activity against all the four bacterial strains. These results were similar to the study conducted by Raja Ratna Reddy et al.,[21] which showed the effect of neem bark extract against bacterial and fungal growth. Ancient Indian civilization has used neem for all types of infections, and the cure basically relies not only on its antibacterial activity but also on its anti-inflammatory properties. A study by Kaur et al.[22] suggested that nimbidin, which is an active constituent of neem bark, suppressed the function of macrophages and neutrophils, thus reducing the inflammation. A study by Okemo et al.[23] showed that neem plant crude extract was very effective against S. aureus and Escherichia More Details coli. Current antimicrobial therapy acts as a bactericidal or bacteriostatic agent without anti-inflammatory property. A report “Neem: A tree for solving global problems.” was published by the US National Academy of Sciences in 1992.[24]A. indica bark extract acts both as antibacterial and anti-inflammatory agent, which makes it an apt choice for the treatment of various bacterial infections. Thus, A. indica has surplus benefits and its role as an antibacterial agent opens the gate for newer scope in the treatment of antibacterial infections with lesser adverse effects.

Conclusion

This study has shown that A. indica bark extract has a potent antibacterial property against various strains of bacterial pathogens. Future research in the isolation of bioactive compounds responsible for antibacterial property can give a new hope in treating bacterial infections with less toxicity.

Acknowledgement

We are grateful to Dr. Bala Murugan Velu, Professor of Microbiology, Sri Lakshmi Narayana Institute of Medical Sciences (SLIMS), Puducherry, India, for his continuous support and encouragement.